CN113278090A

CN113278090A - Albizzia julibrissin polysaccharide, preparation method and application

Info

Publication number: CN113278090A
Application number: CN202110721526.9A
Authority: CN
Inventors: 罗巅辉
Original assignee: Huaqiao University
Current assignee: Huaqiao University
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2021-08-20
Anticipated expiration: 2041-06-28
Also published as: CN113278090B

Abstract

The invention discloses albizia julibrissin polysaccharide, a preparation method and application. The preparation method of the invention is adopted to carry out acid-assisted hot water extraction on the albizia flower polysaccharide. And (3) carrying out ethanol precipitation and repeated freeze thawing on the crude extract to obtain a primary crude extract, deproteinizing the primary crude extract to obtain a secondary crude extract, and purifying the secondary crude extract by DEAE-Sepharose CL-6B and Sephacryl S-400HR column chromatography under specific preparation conditions to obtain a final product. The albizzia julibrissin polysaccharide prepared by the method is polymerized by homogeneous polysaccharide molecules with the molecular mass of 550kDa, consists of arabinose, galactose and galacturonic acid in equal proportion, has the material degradation temperature of 220-340 ℃, and is a high-temperature resistant material. Compared with the traditional hot water extraction, the crude extract obtained by the method has larger sugar content; the finally obtained albizia julibrissin polysaccharide can obviously reduce the relative expression quantity (P <0.05) of mRNA (messenger ribonucleic acid) of TNF-alpha and COX-2 inflammatory factors and iNOS (nitric oxide synthase) genes in RAW264.7 cells induced by LPS (low-cholesterol) and can be used for preparing a medicament for assisting in treating inflammation-related central system diseases.

Description

Albizzia julibrissin polysaccharide, preparation method and application

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to albizia julibrissin polysaccharide, a preparation method and application thereof.

Background

Albizzia julibrissin is Albizia julibrissin belonging to Albizzia, and its dried inflorescence is often used as medicine for tranquilizing, hypnotizing and treating uneasiness. The reports show that the albizia flower water-decocted extract has strong hypnotic effect, but specific components of the compound are not clear.

At present, the research on the albizia julibrissin polysaccharide is less at home and abroad, and the research on the function of the albizia julibrissin polysaccharide is only limited to the research on the antioxidant activity of the crude polysaccharide. Yuanjian plum, etc. (response surface method is preferred and the extraction process of albizia flower polysaccharide [ J ]. pharmacy, 2013,24(23): 2145:. 2148) crude polysaccharide is extracted from albizia flower by using a hot water extraction method, and the polysaccharide extraction rate is optimized by using a response surface method, the optimal extraction process is that the extraction temperature is 91.4 ℃, the extraction time is 3.7h, the liquid-material ratio is 26.6:1(ml/g), and the actual extraction rate of polysaccharide under the condition is 5.79%. Yuanjiamei and the like (extraction process of albizia julibrissin polysaccharides and antioxidant activity research thereof [ J ]. Hubei agricultural science and technology, 2013,52(11):2625 and 2628) use a hot water extraction method and an orthogonal method to optimize the extraction rate of albizia julibrissin polysaccharides, the optimal extraction process is that the extraction temperature is 90 ℃, the extraction time is 4 hours, the liquid-material ratio is 30:1, the extraction is carried out for 2 times, the actual extraction rate of the polysaccharides under the condition is 5.72%, and the obtained crude albizia julibrissin polysaccharides have strong scavenging capacity on hydroxyl radicals and nitrite ions.

The research on the albizia julibrissin polysaccharides in the prior art is only limited to the level of crude polysaccharides, and the polysaccharide components are undefined, so that the preparation method and the structure of the albizia julibrissin polysaccharides need to be deeply researched, and a new object is provided for the development of substitute products of natural drug components without toxic and side effects.

Disclosure of Invention

One of the purposes of the present invention is to provide an albizzia julibrissin, wherein the polysaccharide is a homogeneous heteropolysaccharide, the weight average molecular weight of the heteropolysaccharide is 550kDa, the heteropolysaccharide is composed of arabinose, galactose and galacturonic acid with the molar ratio of 1.10:1.01:1, the glycosidic bond is → 2) -beta-D-GalAp- (1 →, → 2) -beta-D-Galp- (1 → and → 2) -beta-D-Araf- (1 →, and the material degradation temperature is 220-.

The invention also aims to provide the application of the albizia julibrissin polysaccharide in preparing medicines for treating central system diseases related to inflammation.

Preferably, the disease of the central nervous system is alzheimer's disease.

Another object of the present invention is to provide a method for preparing albizia julibrissin polysaccharides, comprising the following steps:

(1) fully drying flower buds of the albizia julibrissin durazz in the sun, extracting by adopting an acid-assisted hot water extraction method, and centrifuging to obtain supernatant, namely a crude extract; the acid-assisted hot water extraction method comprises the steps of extracting materials and extracting solution for 150min in a water bath at 90 ℃ with the mass ratio of 1:65 and the pH value of 1.45;

(2) concentrating the crude extract obtained in the step (1), adding 4 times of volume of absolute ethyl alcohol, standing in a refrigerator at 4 ℃ for 15h, centrifuging, taking the precipitate, dissolving the precipitate with distilled water, repeatedly freezing and thawing for 5 times, and freeze-drying the dissolved solution to obtain a primary crude extract;

(3) dissolving the primary crude extract obtained in the step (2) in distilled water, deproteinizing for 6 times by a Sevag method, adding 4 times of volume of absolute ethyl alcohol, precipitating, and freeze-drying to obtain a secondary crude extract;

(4) dissolving 500mg of the secondary crude extract obtained in the step (3) in 15mL of distilled water, loading the mixture on a DEAE-Sepharose CL-6B column, wherein the elution step is a two-step method, firstly eluting 1500mL of the mixture by the distilled water at the speed of 60mL/h, not collecting the eluent in the step, then using 600mL of the distilled water and 600mL of 1M NaCl solution to carry out linear gradient elution, using an automatic part collector to collect 360mL to 720mL of the effluent, dialyzing the collected effluent for 20h respectively by tap water and the distilled water by using a dialysis bag with the molecular weight cutoff of 3500 and 5000, condensing the dialysate, and freeze-drying to obtain the albizia flower primary polysaccharide;

(5) dissolving 100mg of the primary polysaccharide obtained in the step (4) in 3mL of distilled water, loading the solution on a Sephacryl S-400HR column, using 0.2mol/L NaCl solution as eluent, adjusting the flow rate to be 0.5mL/min, collecting one tube every 24min, collecting 12mL of eluent in each tube, collecting 40-60 tubes of eluent, dialyzing the eluent by a dialysis bag with the molecular weight cutoff of 3500-.

Preferably, the crude extract in step (2) is concentrated by 40-fold under reduced pressure using a rotary evaporator at 60 ℃.

In a preferable mode, the primary crude extract in the step (3) is prepared into a 3% solution, and then protein is removed by using a Sevag method.

Preferably, the gel column in step (4) has a specification of 4.6cm (diameter) × 33cm (height), and the cut-off molecular weight of the dialysis bag is 3500.

Preferably, in the step (5), the Sephacryl S-400HR gel column specification is 2.6cm (diameter) × 85cm (height), and the cut-off molecular weight of the dialysis bag is 3500.

The invention has the beneficial effects that:

(1) the albizia julibrissin polysaccharide prepared by the invention is a novel polysaccharide, and has uniform components and a definite structure. The polysaccharide has characteristic functional groups of carbohydrate substances and is polymerized by homogeneous polysaccharide molecules with the molecular mass of 550kDa, the glycogen consists of arabinose, galactose and galacturonic acid, the molar ratio of the monosaccharides is 1.10:1.01:1, the glycosidic bond is → 2) -beta-D-GalAp- (1 →, → 2) -beta-D-Galp- (1 → and → 2) -beta-D-Araf- (1 →, and the substance degradation temperature is 220-.

(2) The preparation method provided by the invention is used for extracting the albizia julibrissin polysaccharide by an acid-assisted hot water method, and compared with the traditional hot water extraction method, the extraction rate of the polysaccharide obtained by extraction is higher. And carrying out alcohol precipitation and repeated freeze thawing on the crude extract to obtain a primary crude extract, deproteinizing the primary crude extract to obtain a secondary crude extract, and purifying the secondary crude extract by DEAE-Sepharose CL-6B and Sephacryl S-400HR column chromatography under specific preparation conditions to obtain a final product.

(3) The albizzia julibrissin polysaccharide prepared by the invention has a remarkable anti-inflammatory effect, the albizzia julibrissin polysaccharide with the mass concentration of 150 mu g/mL can remarkably reduce the relative expression amount (P <0.05) of mRNA (messenger ribonucleic acid) of TNF-alpha and COX-2 inflammatory factors in RAW264.7 cells induced by LPS, the inhibition rates are 71.31% and 68.14% respectively, the expression amount (P <0.05) of iNOS genes can be remarkably reduced, and the effect is not remarkably different from that of a positive control group (dexamethasone). TNF-alpha is an important factor of brain injury, a large amount of TNF-alpha is released by cells after cerebral hemorrhage, and high expression of TNF-alpha can be a secondary reaction of blood-brain barrier damage, nerve cell apoptosis and the like. Overexpression of the iNOS gene releases a large amount of nitric oxide, thereby exacerbating neuronal cell damage. The albizia julibrissin polysaccharide prepared by the invention can effectively inhibit the over-expression of TNF-alpha and iNOS in inflammatory cells, has no toxic or side effect and definite active ingredients, and can be used as an auxiliary medicine for inflammation-related central system diseases.

Drawings

FIG. 1 is a high performance gel exclusion chromatogram of Albizzia julibrissin AJP of example 3 of the present invention.

Fig. 2 is an infrared spectrum of albizzia julibrissin AJP in example 3 of the present invention.

FIG. 3 is a gas chromatogram of standard monosaccharide and AJP of example 3 of the present invention.

Fig. 4 is a thermogravimetric analysis of albizzia julibrissin AJP of example 3 according to the present invention.

Fig. 5 is a nuclear magnetic map of albizzia julibrissin AJP of example 3 according to the present invention.

A:¹H,B:¹³C,C:COSY,D:HSQC,E:TOCSY,F:NOESY。

FIG. 6 is a transmission electron micrograph of Albizzia julibrissin polysaccharide AJP in example 3 of the present invention.

FIG. 7 is a graph showing the effect of Albizzia julibrissin polysaccharide AJP on LPS stimulation of TNF- α (A), IL-1 β (B), COX-2(C) and iNOS (D) mRNA expression in RAW264.7 mouse macrophages in accordance with example 4 of the present invention.

Detailed Description

The technical solution of the present invention is further illustrated and described by the following detailed description.

Example 1 optimization of acid-assisted Hot Water extraction Process for Albizzia julibrissin polysaccharide extraction

The experimental method comprises the following steps: weighing 1g of flower buds of albizia julibrissin, adding corresponding distilled water according to the experimental design shown in table 1, adjusting the pH value, extracting at a specific extraction temperature to obtain a crude extract of albizia julibrissin, and detecting the polysaccharide extraction rate of the crude extract by adopting a phenol-sulfuric acid method. According to the single-factor experiment result, the influence of the extraction time on the total sugar extraction rate is found to be small, so that the fixed extraction time is 150min, a Box-Behnken model is adopted to arrange the experiment, the influence of the acid-assisted hot water extraction method on the albizia julibrissin polysaccharide extraction rate is researched by taking the pH value, the extraction temperature and the liquid-material ratio as main experiment factors, and the result is shown in the table 1.

TABLE 1 Box-Behnken design scheme and yield for extracting total sugar from flos Albizziae by acid-assisted hot water extraction

As a result: and (3) performing secondary response surface regression fitting on the data in the table 1 by adopting Design-Expert 8.0 software to obtain a model between the polysaccharide extraction rate Y and the inspected 3 factors, and a model significance test result and an R value. The regression equation for this model is Y ═ 6.35+0.37A-0.56B +0.16C-0.11AB +0.06AC +0.1BC-0.81A²-0.54B²-0.89C²Coefficient of multivariate correlation R²The variable accounting for 99.01% can be interpreted using this model, adjusted R0.9901²And predicting R²0.9773 and 0.9030 respectively, the difference is less than 0.1, p is less than 0.0001, the significance is realized, the distortion item is 0.2944, the significance is not obvious, and the model prediction is reliable and accurate. The optimal process conditions for obtaining the acid-assisted hot water extraction albizia polysaccharide by combining the mathematical analysis of the regression model are as follows: the liquid-material ratio is 65:1, the extraction temperature is 90 ℃, the leaching pH is 1.45, the extraction time is 150min, the model prediction value of the total sugar extraction rate is 6.56%, and the verification test value is 6.49%.

Example 2 preparation of Albizzia julibrissin polysaccharide

(1) Fully drying flower buds of albizia julibrissin durazz, weighing 100g of the flower buds, putting the flower buds into 6500mL of distilled water, adjusting the pH value to 1.45 by using hydrochloric acid, extracting the flower buds for 150min in a constant-temperature water bath kettle at 90 ℃, and centrifuging the flower buds for 5min at 3500r/min to obtain a supernatant serving as a crude extraction solution;

(2) and (2) concentrating the crude extract obtained in the step (1) to 150mL by using a 60 ℃ rotary evaporator under reduced pressure, cooling, stirring, slowly adding 600mL of absolute ethyl alcohol, placing in a refrigerator at 4 ℃ for more than 10h, centrifuging at 3500r/min for 10min, and taking a precipitate. Dissolving the precipitate with 100mL of distilled water, freezing at-20 deg.C for 24h, thawing in 60 deg.C water bath, centrifuging to remove impurities to obtain dissolved solution, freezing and thawing the dissolved solution again, freezing and thawing repeatedly for 5 times, and freeze drying the dissolved solution to obtain primary crude extract;

(3) fully dissolving the primary crude extract obtained in the step (2) in distilled water, adding a Sevage reagent (chloroform: n-butyl alcohol is 4:1) according to the total volume of 1/4, fully oscillating for 3 hours, centrifuging to collect an upper-layer aqueous phase dissolved solution, repeating the steps for 6 times on the dissolved solution until an intermediate layer has no solid protein after centrifugation, concentrating the upper-layer dissolved solution, adding 4 times of volume of absolute ethyl alcohol, standing in a refrigerator at 4 ℃ for more than 10 hours, centrifuging to obtain a precipitate, and freeze-drying to obtain a secondary crude extract;

(4) subjecting the second crude extract to primary purification with DEAE-Sepharose CL-6B chromatography column, dialyzing, and freeze drying to obtain primary polysaccharide, which comprises the following steps:

a. equilibration of DEAE-Sepharose CL-6B gel column: and (3) intermittently vacuumizing the gel by using a circulating water type vacuum pump until bubbles do not appear in the gel any more. A small amount of distilled water was added to a 4.6cm diameter glass column, and the gel suspension was slowly drained into the column with a glass rod, which avoided the formation of bubbles. Opening an elution cup and a constant flow pump, adjusting the flow rate to 60mL/h, balancing a chromatographic column with more than 1200mL of distilled water, wherein the balanced chromatographic column is 4.6cm (diameter) multiplied by 33cm (height);

b. loading and collecting: 500mg of the second crude extract was dissolved in 15mL of distilled water, centrifuged, and the supernatant was applied to the above equilibrated DEAE-Sepharose CL-6B column (4.6X 33 cm). And opening the constant flow pump, adjusting the flow rate to be 60mL/h, eluting by using distilled water for more than 1200mL, then respectively filling 600mL of distilled water and 600mL of 1M NaCl solution into the gradient cup, performing linear gradient elution, opening the automatic partial collector to collect effluent, and setting the collector to collect 12mL of effluent in each tube. The sugar content distribution of the collected liquid is measured by a phenol-sulfuric acid method, and 360mL to 720mL of effluent liquid, namely the collected liquid of 30 to 60 pipes, is combined. Dialyzing the sugar-containing collected liquid with a dialysis bag with molecular weight cutoff of 3500, dialyzing with running water for 20h, dialyzing with distilled water for 20h, concentrating the dialyzate by rotary evaporation, and freeze-drying to obtain primary polysaccharide.

(5) Further purifying the primary polysaccharide obtained in the step (4) by using a Sephacryl S-400HR chromatographic column to obtain the albizia flower polysaccharide, wherein the specific steps are as follows:

after air extraction of the Sephacryl S-400HR gel, a column of 2.6cm diameter and 85cm height was prepared and equilibrated with 0.2mol/L NaCl solution. And (3) dissolving 100mg of the primary polysaccharide obtained in the step (4) in 3mL of distilled water, loading the solution on a Sephacryl S-400HR chromatographic column, eluting the solution by using 0.2mol/L NaCl solution at the flow rate of 0.5mL/min, setting that one tube is collected every 24min and each tube is 12mL, collecting 100 tubes by using an automatic part collector, collecting 40-60 tubes of eluent, and dialyzing, concentrating, freeze-drying to obtain the albizia flower polysaccharide which is named as AJP.

Example 3 structural analysis of Albizzia julibrissin polysaccharides

The experimental method comprises the following steps: 1. homogeneity and molecular weight analysis: the homogeneity of the components of sample AJP was identified by High Performance Size Exclusion Chromatography (HPSEC) using a supersar ks-804 column with a mobile phase of ultra pure water at a flow rate of 1mL/min and a column temperature of 50 ℃ and a differential refractometer detector (RIU). A molecular weight standard curve was prepared based on the peak time of dextran standards on the sugar ks-804 column, and the relative molecular mass of AJP was calculated. Taking 0.5-1mg/mL AJP aqueous solution to perform full-spectrum scanning (190-800nm), and detecting the purity of the sample.

2. Physical and chemical property analysis: the samples were analyzed for compound type using infrared spectroscopy (FTIR) and 2mg of sample AJP was subjected to KBr pellet, Nicolet Nexus 470 type infrared spectrometer. AJP thermal property analysis of sample, AJP completely dried was put in a 70 μ L alumina crucible, and detected at 50-600 ℃ by a synchronous thermal analyzer at a temperature rising rate of 10K/min with nitrogen as a carrier gas. To analyze the monosaccharide composition of AJP, 30mg of AJP was hydrolyzed with 1mol/L sulfuric acid at 100 ℃ for 8h, the hydrolysate was neutralized to neutrality with barium carbonate, and the supernatant was freeze-dried to obtain a monosaccharide hydrolysate. After derivatization of the hydrolyzate by trimethylsilylation, the derivatives were analyzed by gas chromatography: the hp-5 capillary chromatographic column is programmed with a temperature of 160 ℃→ 180 ℃ (20 ℃/min) → 220 ℃ (8 ℃/min, hold 2min) → 250 ℃ (2min), and a detector temperature of 280 ℃.

3. Nuclear magnetic resonance analysis: weighing thoroughly dried sugar sample 20mg with 1mLD₂Dissolving O, freeze drying, exchanging for 3 times, and replacing active hydrogen with deuterium. With 0.7mLD₂After the O is dissolved, the O is placed in a nuclear magnetic tube,¹HNMR and¹³CNMR spectra were measured at 25 ℃ under 500MHz and 125MHz conditions using an AVANCE-500MHz NMR spectrometer, respectively.¹Chemical shifts of HNMR were referenced to a HOD signal of delta 4.7ppm at 25 ℃. The 2DNMR spectra were generated using standard Bruker software, including¹H/¹H coreference spectrum (¹H-¹H COSY), total correlation spectrum (TOCSY), heteronuclear single quantum correlation spectrum (HSQC), and nuclear proton precession effect enhancement spectrum (NOESY).

4. Observation by a transmission electron microscope: preparing a sugar sample into a 1mg/mL solution, adding an SDS solution according to a weight ratio of 1:1, dissolving in water at 80 ℃ for 2h, gradually diluting the solution to 5 mu g/mL, then carrying out water bath at 80 ℃ for 2h, and continuously stirring. A drop of the filtrate was dropped onto a porous carbon film (200 mesh, mesoscopic) supported by a copper mesh, naturally dried in the air, and the morphology of the molecule was observed with a transmission electron microscope (voltage 80 KV).

The experimental results are as follows: the results of high performance size exclusion chromatography of albizzia julibrissin AJP are shown in fig. 1. The result showed that only 1 peak appeared at 5.945min, which was sharp and symmetrical, indicating AJP with good homogeneity and single component. The material composition of purified sample AJP can be determined using FTIR detection of characteristic functional groups, as shown in FIG. 2. As can be seen, at 3410cm^-1，2900cm^-1，1640cm^-1And 1050cm^-14 absorption peaks, namely-OH stretching vibration absorption peak, C-H stretching vibration peak, C ═ O absorption peak and C-O-C vibration peak, respectively appear, which represent characteristic functional groups of the carbohydrate, and the specification AJP indicates that the carbohydrate is the carbohydrate. According to the glucose standard curve (y ═ 0.0174x +0.01145, R²0.999), a sugar content of AJP was calculated to be 98.10 ± 2.03%. AJP contains no protein and nucleic acid substances through 190-800nm full-wave spectrum scanning. Preparing a molecular mass standard curve according to the standard glucan to obtain an equation of-4 x +6.31 (R)²0.99), a molecular mass of AJP was calculated to be 550kDa based on HPSEC peak time of AJP, 5.945 min.

Polysaccharide AJP was completely hydrolyzed with sulfuric acid, and the hydrolysate was derivatized by trimethylsilylation, and the derivative was analyzed by GC to obtain AJP monosaccharide composition, as shown in fig. 3. According to the GC results of the standard monosaccharide derivative, AJP consisted of arabinose, galactose and galacturonic acid in a molar ratio of 1.10:1.01: 1. AJP the results of the thermogravimetric analysis (TGA) are shown in FIG. 4. The DTG curve is the first derivative of the TGA curve, making the results more intuitive. As can be seen from the figure, the first weight loss step occurs within 50-150 ℃, mainly the evaporation of adsorbed water, with a small loss of sugar-like. The second step occurs in the range of 220-340 c with a weight loss of over 50% due to the severe decomposition and depolymerization of the structure at AJP at this temperature.

TABLE 2 AJP¹H and¹³chemical shift of C

FIG. 5 is a nuclear magnetic resonance map of AJP, obtained AJP from the nuclear magnetic resonance map¹H and¹³chemical shifts of C are shown in Table 2. From the chemical shifts shown in Table 2, it was found that AJP consisted of → 2) - β -D-GalAp- (1 → 2) - β -D-Galp- (1 → and → 2) - β -D-Araf- (1 → glycosidic bond. FIG. 6 is a transmission electron micrograph of AJP showing that AJP is a polysaccharide with an unbranched structure.

Example 4 cytotoxicity assay and anti-inflammatory Effect study

The experimental method comprises the following steps: the cells (RAW264.7) were cultured in DMEM medium containing 10% serum and 1% double antibody. Culturing at 37 deg.C in 5% carbon dioxide incubator for about 48h, and subculturing with cell scraper. After culturing the cells in the 96-well plate, AJP was tested for cytotoxicity on RAW264.7 using MTT method, setting six AJP mass concentrations of 600, 300, 150, 75, 25 and 1 μ g/mL. Lipopolysaccharide (LPS) with the concentration of 1 mu g/mL is used for inducing RAW264.7 cells to generate inflammation, and an inflammation model is established. Experimental arrangement blank, negative control (LPS), positive control (dexamethasone DXMS) and sample (75 μ g/mL AJP and 150 μ g/mL AJP) groups were set up in 3 replicates per group. After RAW264.7 cells are cultured in the 6-well plate for 24h, the culture medium corresponding to each group is replaced to continue culturing for 24 h. Total RNA was extracted from each group of cells according to Eastep Super total RNA extraction kit (Promega corporation), reverse transcription of cDNA was performed according to the reverse transcription kit, and real-time fluorescent quantitative PCR was performed using qPCR Master Mix kit (Promega corporation) under amplification conditions of 95 ℃ for 15sec, 60 ℃ for 30sec, and 72 ℃ for 30sec (45 cycles). With housekeeping gene GAPDH as reference gene, 2^-ΔΔCTMethod for calculating TNF-alpha, IL-6, COX-2 and iNOSRelative expression amount of mRNA.

The experimental results are as follows: to investigate the anti-inflammatory effect of AJP, its non-toxic incubation concentration on RAW264.7 cells was first sought to ensure the accuracy of the subsequent anti-inflammatory experiments. The experimental results showed that the addition of AJP at 75. mu.g/mL or 150. mu.g/mL did not cytotoxicity the growth of RAW264.7 cells, so 2 concentrations were selected for subsequent studies. To establish an inflammation model, 1. mu.g/mL lipopolysaccharide was added into RAW264.7 cells and used as a negative control (LPS). As shown in FIG. 7, the relative expression levels of the mRNAs of TNF- α (P <0.0001), IL-1 β (P <0.0001), COX-2(P <0.0001), and iNOS (P <0.05) in the negative control group were significantly increased compared to the blank group without lipopolysaccharide, indicating that several inflammation models were successfully constructed. A positive control group (DXMS) is set, 75 mu g/mL dexamethasone is added to incubate RAW264.7 cells together with lipopolysaccharide, a sample group comprises AJP with mass concentrations of 75 mu g/mL and 150 mu g/mL, and anti-inflammatory results are shown in a figure 7. The positive control group and the sample group can obviously reduce the mRNA relative expression quantity (P <0.001) of the inflammatory factor TNF-alpha, and the inhibition rates of the 75 and 150 mu g/mL sample groups are 86.77% and 71.31%, respectively. The significant analysis shows that the inhibition rate of TNF-alpha expression of the two sample groups is not significantly different from that of the positive control group (P > 0.05). AJP has no significant inhibition effect on the relative expression quantity of IL-1 beta mRNA. AJP with 75 and 150 mu g/mL can remarkably reduce the relative expression quantity of COX-2 mRNA (P <0.01) in RAW264.7 cells induced by lipopolysaccharide, and the inhibition rate is 75.38% and 68.14% respectively. 75 mu g/mL of AJP can not reduce the expression level of iNOS genes in inflammatory cells, 150 mu g/mL of AJP can obviously reduce the relative expression quantity of iNOS mRNA (P <0.05), the inhibition rate is 27.99%, and the inhibition effect of the AJP is not obviously different from that of a positive control group (P < 0.187).

The over-expression of inflammatory factors is associated with many central system diseases, such as brain hemorrhage, and the cells release toxic substances such as chemotactic factors, inflammatory factors and nitric oxide, thereby aggravating the damage to neurons. Increased levels of chemokines (TNF-alpha) and inflammatory factors are present in patients with Alzheimer's Disease (AD), and TNF-alpha is neurotoxic and also increases plaque deposition. Through the research, the albizia julibrissin durazzini polysaccharide can obviously reduce the relative expression quantity (P <0.001) of mRNA (messenger ribonucleic acid) of TNF-alpha, COX-2 and iNOS inflammatory factors in RAW264.7 cells induced by LPS (low cholesterol) and has the effect of protecting neurons.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims

1. The albizzia julibrissin polysaccharide is a homogeneous heteropolysaccharide with the weight-average molecular weight of 550kDa, and consists of arabinose, galactose and galacturonic acid in the molar ratio of 1.10:1.01:1, wherein the glycosidic bond consists of → 2) -beta-D-GalAp- (1 →, → 2) -beta-D-Galp- (1 → and → 2) -beta-D-Araf- (1 →, and the material degradation temperature is 220-.

2. Use of the albizzia julibrissin polysaccharide according to claim 1 for the preparation of a medicament for the treatment of inflammation-related central system diseases.

3. The use according to claim 2, wherein the disease of the central nervous system is alzheimer's disease.

4. A preparation method of albizzia julibrissin polysaccharides comprises the following steps:

(4) dissolving 500mg of the secondary crude extract obtained in the step (3) in 15mL of distilled water, loading the mixture on a DEAE-Sepharose CL-6B column, wherein the elution step is a two-step method, firstly eluting 1500mL of the mixture by the distilled water at the speed of 60mL/h, not collecting the eluent in the step, then using 600mL of the distilled water and 600mL of 1M NaCl solution to carry out linear gradient elution, using an automatic part collector to collect 360mL to 720mL of the effluent, dialyzing the collected effluent by a dialysis bag with the molecular weight cutoff of 3500-;

5. The method for preparing albizzia julibrissin durazzini polysaccharide as claimed in claim 4, characterized in that: and (3) decompressing and concentrating the crude extract in the step (2) by 40 times by using a rotary evaporator at 60 ℃.

6. The method for preparing albizzia julibrissin durazzini polysaccharide as claimed in claim 4, characterized in that: the specification of the gel column in the step (4) is 4.6cm in diameter and 33cm in height, and the cut-off molecular weight of the dialysis bag is 3500.

7. The method for preparing albizzia julibrissin durazzini polysaccharide as claimed in claim 4, characterized in that: in the step (5), the specification of the Sephacryl S-400HR column is 2.6cm in diameter and 85cm in height, and the cut-off molecular weight of the dialysis bag is 3500.

8. Albizzia julibrissin polysaccharide prepared by the preparation method according to any one of claims 4-7.